Active human Cytomegalovirus infection and Glycoprotein B genotypes in Brazilian pediatric renal or hematopoietic stem cell transplantation patients

A prospective analysis of active Human Cytomegalovirus infection (HCMV) was conducted on 33 pediatric renal or hematopoietic stem cell post-transplant patients. The HCMV-DNA positive samples were evaluated for the prevalence of different gB subtypes and their subsequent correlation with clinical signs. The surveillance of HCMV active infection was based on the monitoring of antigenemia (AGM) and on a nested polymerase chain reaction (N-PCR) for the detection of HCMV in the patients studied. Using restriction analysis of the gB gene sequence by PCR-RFLP (Restriction Fragment Length Polymorphism), different HCMV strains could be detected and classified in at least four HCMV genotypes. Thirty-three pediatric recipients of renal or bone marrow transplantation were monitored. Twenty out of thirty-three (60.6%) patients demonstrated active HCMV infection. gB1 and gB2 genotypes were more frequent in this population. In this study, we observed that gB2 had correlation with reactivation of HCMV infection and that patients with mixture of genotypes did not show any symptoms of HCMV disease. Future studies has been made to confirm this.505

Skin neuro-immuno-endocrinology response related to photoaging : in vitro and clinical assessment of a new compound as therapeutical alternative in dermatology

Orientador: Mary Luci de Souza QueirozTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: O envelhecimento cutâneo é resultado de dois processos independentes, clinica e biologicamente distintos, que afetam a pele simultaneamente. Ao primeiro, conhecido como "relógio biológico", damos o nome de envelhecimento intrínseco, o qual afeta a pele na mesma proporção em que órgãos internos são afetados, como por exemplo, a lenta e irreversível degeneração tecidual. O segundo, denominado fotoenvelhecimento, é resultado da exposição da pele a agentes agressores ambientais, primeiramente radiação ultravioleta (RUV). Sobre o impacto da RUV, especialmente RUVA e RUVB, a pele passa por inúmeras alterações, incluindo hiperpigmentação, fotodano, alterações histológicas e imunológicas. Tendo em vista a complexidade do fotoenvelhecimento, podemos dividir este trabalho em duas partes, onde avaliamos em uma primeira etapa os efeitos da RUV sobre a resposta neuroimuno-endocrinológica da pele através da mensuração de parâmetros específicos em culturas de fibroblastos e queratinocitos humanos. Após a realização dos experimentos propostos para a primeira parte deste trabalho, passamos a considerar para a segunda etapa, a possível ação in vitro e in vivo do extrato de Rhodiola rosea (ERR) em associação com o dipeptídeo Lcarnosina (LC) sobre a reversão ou controle dos efeitos deletérios da RUV sobre a pele. Nossos resultados demonstram que durante a foto-exposição, culturas celulares in vitro apresentaram importantes alterações nos parâmetros avaliados, especialmente após a exposição crônica, dentre elas: redução na produção de IL-1a, TNF-a e IFN-g, redução nos níveis de b-endorfina e encefalina, aumento na atividade de metaloproteinases (MMPs), inibição na atividade do inibidor tecidual de metaloproteinase 1 (TIMP-1), redução nos níveis de colágeno e elastina e redução na atividade das enzimas superóxido dismutase (SOD) e catalase (CAT). No perfil de resposta das células tratadas com ERR, pudemos observar uma importante função adaptógena do extrato, onde na maioria dos parâmetros, constatamos uma modulação da resposta celular de forma dose dependente. Os resultados obtidos com L-carnosina foram importantes, especialmente no que diz respeito aos níveis de colágeno, elastina e atividade das enzimas antioxidantes. Quando o perfil de resposta da associação entre ambos (ARRLC) é observado, pode-se perceber que, em alguns parâmetros, não houve uma potencialização dos efeitos. No entanto, dada a complexidade da resposta neuro-imuno-endocrinológica da pele relacionada ao fotoenvelhecimento, pode-se concluir que a utilização de compostos com mecanismos de ação distintos é imprescindível, uma vez que diferentes processos metabólicos levam ao dano tecidual e à aparência estética comprometida da pele fotoenvelhecida. Para a realização dos estudos clínicos, uma emulsão placebo ou emulsão contendo 1% de ARRLC foi utilizada pelas voluntárias em dois estudos independentes, sendo que o primeiro foi realizado durante 28 dias para avaliação dos parâmetros subjetivos - conforto da pele, redução da sensação de desconforto após Stinging-Test (ST) e melhora do ressecamento da pele. O segundo estudo foi realizado durante 56 dias, onde avaliou-se em D1, D28 e D56, a melhora do relevo cutâneo e a redução da perda de água transepidérmica. Os resultados obtidos demonstram que aproximadamente 90% das voluntárias reportaram uma melhora do conforto da pele e 75% reportaram redução do ressecamento. Além disso, a utilização da emulsão contendo 1% de ARRLC apresentou uma redução de aproximadamente 36% na sensação de desconforto após ST. Além disso, o tratamento com a emulsão contendo 1% de ARRLC resultou em redução considerável da perda de água transepidérmica e na melhora do relevo cutâneo. Estes resultados clínicos, associados aos resultados obtidos in vitro, nos encorajam a sugerir o uso de ARRLC como alternativa terapêutica no tratamento tópico do fotoenvelhecimento.Abstract: Cutaneous ageing is a complex biological phenomenon consisting of two components; intrinsic ageing, which is largely genetically determined and extrinsic ageing caused by environmental exposure, primarily UV light. Under the impact of ultraviolet radiation (UVR), skin undergoes several changes, including enhanced pigmentation, photodamage and immunosuppression. Because photoaging culminates in important alterations in skin function and appearance, we can separate this work in two different phases. In the first phase, we evaluated the effects of ultraviolet radiation (UVR) on skin neuro-immuneendocrinological response through the measurement of specific parameters in fibroblasts and keratinocytes human cell cultures. After the conclusion of experiments proposed for the first phase of this work, the possible in vitro and in vivo effects of Rhodiola rosea extract (ERR) in combination to di-peptide Lcarnosine (LC) in the control of deleterious effects of UVR exposure on the skin were evaluated. Our results demonstrated that during the UVR, the cell cultures promoted crucial alterations in the parameters evaluated, mainly after chronic exposure. The alterations observed were: the reduction in the production of IL-1a, TNF-a, IFN-g, b-endorphin and enkephalin, reduction in the levels of matrix metalloproteinase tissue inhibitor-1, increase of metalloproteinase activity, reduction of collagen and elastin production and inhibition of the antioxidant enzymes superoxide dismutase and catalase. In the cell groups that received ERR, we observed an important adaptogenic function of the extract, because in the majority of the parameters, it was able to modulate the cell response in a dose-dependent manner. The results obtained in the LC treated groups, we could also observe important effects, mainly in collagen and elastin synthesis and in the antioxidant enzymes activities. When we consider the response obtained with the association of both ERR and LC (ARRLC), we could see that it is not able to enhance the effects obtained with ERR or LC separately. Therefore, due to the complexity of the neuro-immune-endocrinological response during the photoaging, we can conclude that the use of compounds with different mechanisms of action is crucial, because distinct metabolic process lead to tissue damage and terrible appearance of photodamaged skin. For the clinical trials, a placebo emulsion or a emulsion containing 1% of ARRLC were used by the volunteers in two independent studies, in which the first one was performed during 28 days for evaluation of subjective parameters, including skin comfort, reduction of skin dryness sensation and reduction of skin discomfort sensation after Stinging-Test (ST). The second study was performed during 56-days and the evaluations were carried out on D1, D28 and D56. The parameters evaluated were the improvement of cutaneous relief and the reduction of transepidermal water loss. The obtained results demonstrated that approximately 90% of the volunteers perceived an increase in skin comfort and 75% reported reduction of skin dryness sensation. In addition, the emulsion containing 1% of ARRLC was able to reduce in approximately 36% the sensation of discomfort after ST. These clinical results and the in vitro findings, encourage us to suggest the use of ARRLC as alternative ingredient in the topical treatment of skin photoaging.DoutoradoDoutor em Farmacologi

Avaliação dos efeitos imuno-hematopoieticos da geleia real em modelo experimental de listeriose murina

Orientador: Claudia Brincoletto TrindadeDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Avaliamos, neste trabalho, os efeitos da Geléia Real (GR) sobre o crescimento e diferenciação dos precursores hematopoiéticos da medula óssea e baço em animais infectados com Listeria monocytogenes. Além disso, a atividade estimuladora de colônias do soro, as alterações no peso do baço e a resistência dos animais frente a infecção também foram avaliados. Para tanto, camundongos BALB/c foram tratados profilaticamente, por um período de 7 dias consecutivos, com GR nas doses de 12S, 2S0, SOOe 7SO mg/Kg, por gavagem. Posteriormente, os animais foram infectados intraperitonealmente com uma dose subletal (1,Sx103 bactérias/animal) de Listeria monocytogenes. Para avaliarmos a resistência dos animais frente à infecção, realizamos uma curva de sobrevida utilizando uma dose letal desta bactéria (1,Sx105 bactérias/animal). Nossos resultados demonstraram um decréscimo significativo no número de precursores hematopoiéticos da medula óssea nos animais infectados com Listeria monocytogenes, além de uma significativa hematopoiése extramedular. No entanto, quando os animais foram tratados profilaticamente com GR, independentemente da dose administrada, houve uma reversão da mielossupressão induzida pela infecção, resultando em níveis normais de CFUGM. Outro aspecto importante é com relação à produção de fatores estimuladores de colônias de células hematopoiéticas. Neste sentido, observamos que os animais infectados apresentaram uma maior atividade estimuladora de colônias no soro. A administração prévia de GR, nas quatro doses avaliadas, nos animais infectados, aumentou significativamente a atividade estimuladora do soro, resultando em um aumento no número de CFU-GM em relação aos grupos apenas infectados. Além disso, observamos também, um aumento do peso do baço dos animais infectados, enquanto que valores normais foram obtidos quando os animais receberam GR antes da infecção, em todas as doses administradas. o tratamento com GR aumentou a resistência dos animais frente à listeriose, sendo que a probabilidade de sobrevida para as doses de 125, 250, 500 e 750 mg/Kg foi de 37,5, 37,5, 25 e 12,5%, respectivamente, não havendo diferenças estatisticamente significativas entre si. Nossos resultados sugerem que a GR possui uma potente atividade imunomoduladora, corroborando com resultados existentes na literatura e anteriormente obtidos em nosso laboratório. A ampla faixa terapêutica do composto frente à listeriose foi comprovada, visto que não houve uma alteração significativa entre as respostas imuno-hematopoiéticas nas doses avaliadas, sugerindo que mesmo em baixas concentrações, a GR é capaz de restaurar o sistema imunológico frente à infecçãoAbstract: We evaluated in this work the effects of Royal Jelly (RJ) on growth and differentiation of bone marrow and spleen hematopoietics progenitors in Listeria monocytogenes (LM) infected mice. We also evaluated serum colonystimulating activity, alterations in spleen weight and animal resistance front to the infection. In such way, BALB/c mice were profilacticly treated with RJ by gavage for 7 consecutive days with doses at 125, 250, 500 and 750 mg/Kg. Three hours after the last treating day the animais were infected intraperitoneally with a sublethal dose of the bacteria (1x103 LM/animal). To evaluate animal resistance front to the infection we carried out a survival analysis with a lethal dose of the bacteria (1x105 LM/animal). Our results demonstrated significant decrease on the hematopoietic progenitor numbers in LM bearing mice and significant extramedullar hematopoiesis as consequence. In the other hand, myelossupression induced by listeriosis was reverted when bearing mice were treated profilacticly with ali doses of RJ, resulting in normal levels of CFU-GM. Another important aspect regards colony-stimulating factor production by hematopoietic cells. In this direction, we observed that the infected animais presented higher serum colony-stimulating activity. Previous administration of RJ at ali doses in infected mice significantly increased serum stimulatory activity as well as CFU-GM number when compared to non-treated bearing groups. We also observed spleen weight increase in the non-treated infected group at ali doses, which contrasted with normal values obtained in previously treated bearing mice. In addition, the administration of RJ at 125, 250, 500 and 750 mg/Kg increased animal resistance front to listeriosis to 37,5%, 37,5%, 25% and 12,5%, respectively. Our results have demonstrated that RJ has a stimulating effect on the modulation of the immuno-hematopoietic response in mice infected with LM. Moreover, the treatment with RJ increased survival rate in infected mice with lethal dose of the bacteria. We demonstrated that RJ presents extensive therapeutic dose range since the adminisitration of ali evaluated doses (125, 250, 500 and 750 mg/Kg) presented similar resultsMestradoMestre em Farmacologi

Effect of green Coffea arabica L. seed oil on extracellular matrix components and water-channel expression in in vitro and ex vivo human skin models

Background Green Coffea arabica L. seed oil is being widely used in cosmetic formulations, although its effects on human skin cells are not clear and most observations are unpublished. Aims In this study, we evaluated the in vitro effects of green coffee (C. arabica L.) oil (GCO) on the synthesis of collagen, elastin, and glycosaminoglycans (GAG) and in the release of transforming growth factor-beta 1 (TGF-beta 1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human skin fibroblasts. We also investigated the ability of GCO to increase aquaglycerolporins-3 (AQP-3) mRNA expression in cultured keratinocytes and human skin explants.Methods Human fibroblasts were incubated for 48 h with several GCO concentrations (3.12, 6.25, 12.5, 25.0 and 50.0 mg/mL). The levels of growth factors and extracellular matrix compounds in the culture supernatant were measured using commercial kits. To evaluate AQP-3 relative expression, using real-time reverse transcription polymerase chain reaction, keratinocytes were incubated for 3-6 h with the GCO optimal concentration of 25.0 mg/mL. Histological sections of human skin were also incubated with GCO (25.0 mg/mL) and immunostained by antiserum against AQP-3.Results Our results demonstrated that incubation with GCO produces a dose-dependent stimulation in the synthesis of collagen, elastin, and GAG, in addition to increasing the release of the growth factors TGF-beta 1 and GM-CSF. GCO also induced the expression of AQP-3 mRNA, which reached levels up to 6.5-fold higher than those of the control cultures.Conclusion The findings presented herein suggest that GCO might improve physiological balance in the skin, thus allowing the formation of new connective tissue, and preventing epidermis dryness by increasing AQP-3 levels. Taking into account the limitations of in vitro studies, it is encouraging in this context to consider CGO as an adjuvant to be used in dermocosmetic formulations. Clinical studies are in progress in our laboratory aiming to further investigate the protective effects of CGO in the skin.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Expression of differential genes involved in the maintenance of water balance in human skin by Piptadenia colubrina extract

Background Hydration and integrity of the stratum corneum (SC) is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function. The presence of aquaglyceroporins and envelope proteins are crucial to provide greater corneocyte cohesion to keep water and other moisturizers in the skin.Aims In this study, we evaluated the ability of Piptadenia colubrina, a plant native of South American rain forests, in the expression of genes involved in skin capacitance and SC integrity.Methods The expression of genes for aquaporin-3 (AQP3), loricrin, involucrin (INV), and filaggrin (FLG) was measured by real-time PCR, using an in vitro model of human keratinocytes incubated with concentrations of 2.5, 5, 10, and 20 mg/mL of a hydroglycolic extract of P. colubrina (HEPC). The amount of AQP3 protein was also tested by immunohistochemistry in human skin explants. Clinical trials were conducted to evaluate the effects of a gel-cream containing HEPC on the glycerol index and skin capacitance.Results Hydroglycolic extract of P. colubrina increased both the expression and immunoreactivity of AQP3 in cultured keratinocytes and human skin explants. The gene induction to envelope proteins FLG and INV was also observed after cell incubation with HEPC. Skin capacitance was significantly improved in human volunteers under treatment with HEPC-containing cream.Conclusions The extract of P. colubrina promotes cellular hydration and induces gene expression of envelope proteins providing greater corneocyte cohesion to keep water and other moisturizers in the skin and an appropriate epidermal adhesion. The in vitro findings were clinically confirmed and encourage the clinical use of this compound in skin care products.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Expression Of Differential Genes Involved In The Maintenance Of Water Balance In Human Skin By Piptadenia Colubrina Extract.

Hydration and integrity of the stratum corneum (SC) is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function. The presence of aquaglyceroporins and envelope proteins are crucial to provide greater corneocyte cohesion to keep water and other moisturizers in the skin. In this study, we evaluated the ability of Piptadenia colubrina, a plant native of South American rain forests, in the expression of genes involved in skin capacitance and SC integrity. The expression of genes for aquaporin-3 (AQP3), loricrin, involucrin (INV), and filaggrin (FLG) was measured by real-time PCR, using an in vitro model of human keratinocytes incubated with concentrations of 2.5, 5, 10, and 20 mg/mL of a hydroglycolic extract of P. colubrina (HEPC). The amount of AQP3 protein was also tested by immunohistochemistry in human skin explants. Clinical trials were conducted to evaluate the effects of a gel-cream containing HEPC on the glycerol index and skin capacitance. Hydroglycolic extract of P. colubrina increased both the expression and immunoreactivity of AQP3 in cultured keratinocytes and human skin explants. The gene induction to envelope proteins FLG and INV was also observed after cell incubation with HEPC. Skin capacitance was significantly improved in human volunteers under treatment with HEPC-containing cream. The extract of P. colubrina promotes cellular hydration and induces gene expression of envelope proteins providing greater corneocyte cohesion to keep water and other moisturizers in the skin and an appropriate epidermal adhesion. The in vitro findings were clinically confirmed and encourage the clinical use of this compound in skin care products.935-4